Genetically engineered (GE) pigs can be a great model in biomedical research as pigs are physiologically, anatomically, and immunologically similar to humans. However, the main shortcoming of the GE pigs is poor efficiency in their production. Conventionally, GE pigs with specific genetic modification are produced by first generating GE somatic cells then followed by somatic nuclear transfer (SCNT). However, these processes are labor intensive and inefficient. Recent advancement in meganucleases allows us to generate genetic modifications at a higher efficiency and even during embryo development with an optimized approach thus by- passing the necessity of SCNT. Among different meganucleases, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system demonstrates the most appealing results as it is easy to design and utilize. The objective of this project is to optimize utilization of homology directed repair (HDR) during CRISPR/Cas9 mediated gene targeting system in porcine embryos by influencing the activity of NHEJ or HDR. For the proof-of-concept, our target genes for this study are beta-2-microglobulin (B2M) and CD8A as mutations in the genes are likely to result in the loss of functional CD8+ T cells, one of the key components of anti-viral immunity. Our hypothesis is that CRISPR/Cas9 system can successfully induce genetic mutation in the B2M and CD8A genes through HDR during porcine early embryogenesis. The GE pigs without functional CD8+ T cells can serve as an animal model system for the elucidation of mechanisms of pathogenesis and protective immunity in a wide array of viral infectious diseases in future studies. We will address our hypothesis by two specific aims: Aim 1. Stimulation of HDR pathway during CRISPR/Cas9 mediated gene targeting in porcine embryos. Aim 2. Production and characterization of CD8+ T cell deficient pigs. We will characterize the pigs by immune phenotyping and test the utility of the GE pigs in the study of mechanisms of human norovirus vaccine-induced protective immunity using the well-established gnotobiotic pig model system of human norovirus GII.4 infection and diarrhea. Our long-term goal is to establish effective and efficient approach to generate specific mutations through HDR utilizing the CRISPR/Cas9 system during embryogenesis thus increase production efficiency of GE pigs to meet the increasing need of GE pigs in biomedical research. Because of similarities in their physiology to human, pigs have been widely used in biomedicine research such as infectious diseases, immunology, transplantation, cardiovascular disease, and regenerative medicine. The ability to effectively and quickly generate GE pigs will further enhance the value of the pig for dissecting disease mechanisms and validating clinical therapies.